Team:Goettingen/week21-2

V09_17

• Experiment:
  In order to clone the flhDC and FliC into pSB1C3, all components were digested with EcoRI and PstI according to the protocol. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. After measuring the concentration of the purified fragments, an overnight ligation was initiated.
  

• Experiment:
  We prepared overnight cultures of different promoter-gene constructs in two different E. coli strains (MG1655 & BL21) in order to test wether the motility is influenced. The following constructs were used:
  
  - 20E_flhDC
  - 18C_flhDC
  - 20I_flhdc
  - 20E_motA
  - 18C_motA
  - 20E_motB
  - 18C_motB
  

V09_18

• Experiment: For the motility assay the over night cultures, with different constructs, were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find HERE
  

• Experiment:
  The genes of interest, motA, motB and yhjH were digested with XbaI and PstI. The plasmids with the different promoters were treated with SpeI and PstI. The digestion was then controlled via gel-electrophoresis.
  
• Observation and Results:
  Since for all clones band of the expected size could be obtained these were isolated from the gel and purified using PeqGOLD Gelextraction Kit (Peqlab) according to the manual.
  

• Experiment:
  motA, motB and yhjH were ligated into the plasmid pSB1C3 with different promoters (20E, 20I and 18C) according to the protocol.
  

• Experiment:
  Chemical transformation was done according to the standard protocol. The following constructs were used:
  
  - pSB1C3-FliC into E. coli (DH10B)
  - pSB1C3-flhDC into E. coli (DH10B)
  - PSB1C3-RFP into E. coli (MG1655)
  
  
• Observations & Results:
  On all plates only a few colonies were observed.

V09_19

• Experiment: For the motility assay the cultures with different constructs were grown for 6 hours. After that they were spun down and dropped onto M9- or 1% trypton-agar. The exact protocol you can find HERE
  The Following constructs in the E. colistrain MG1655 were used:
  
  -pSB1C3-20I-flhDC
  -pSB1C3-18C-flhDC
  -pSB1C3-20E-flhDC
  -pSB1C3-RFP
  
  
• Observation and Results:
  At the following day (20th of september) the colonies still looked exactly the same. Neither chemotaxis nor swimming could be observed.
  

V09_20

• Experiment:
  Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual. All constructs were digested with XbaI and SpeI conducted as described in the protocol.
  
  - pSB1C3-flhDC
  - pSB1C3-fliC
  
  
• Observation and Results:
  The pSB1C3-flhDC / fliC clones do not host the correctly inserted gene.
  

• Experiment:
  In order to get more fliC, the already quikchanged fliC was amplified via PCR. The PCR product was analyzed via gel-electrophoresis and subsequently purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.
  
• Observation and Results:
  The PCR was not succesful, we could not observe a band at the expected size.
  

• Experiment:
  In order to clone the flhDC into pSB1C3, all components were digested with EcoRI and PstI according to the protocol. The prodcuts were seperated via gel electrophoresis and purified using PeqGOLD Gelextraction Kit (Peqlab).
  

• Experiment:
  Over night cultures were prepared of three colonies of each constructs in order to isolate the plasmid and check the correct insert.
  
  - pSB1C3-18C-motA (DH10B)
  - pSB1C3-18C-motB (DH10B)
  - pSB1C3-18C-yhjH (DH10B)
  - pSB1C3-20E-motA (DH10B)
  - pSB1C3-20E-motA (DH10B)
  - pSB1C3-20E-motA (DH10B)
  - pSB1C3-20I-motA (DH10B)
  - pSB1C3-20I-motB (DH10B)
  - pSB1C3-20I-yhjH (DH10B)
  - pSB1C3-RFP (BL21)
  
  

V09_21

• Experiment:
  The restriction products were separated on a gel in order to verify the digestions success and purify the desired fragments. After ligation of the genes into pSB1C3, we transformed the ligated plasmids into E. coli DH10B according to the protocol.
  
  

• Experiment:
  Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual. All constructs were digested with XbaI and SpeI conducted as described in the protocol.
  
  - pSB1C3-18C-motA (DH10B)
  - pSB1C3-18C-motB (DH10B)
  - pSB1C3-18C-yhjH (DH10B)
  - pSB1C3-20E-motA (DH10B)
  - pSB1C3-20E-motA (DH10B)
  - pSB1C3-20E-motA (DH10B)
  - pSB1C3-20I-motA (DH10B)
  - pSB1C3-20I-motB (DH10B)
  - pSB1C3-20I-yhjH (DH10B)
  - pSB1C3-RFP (BL21)
  
  
• Observation and Results:
  All clones host the correctly inserted gene in the plasmid.